Natural killer (NK) cells play a recently recognized and critical role in innate immunity, functioning through an array of stimulatory and inhibitory cell-surface receptors that mediate recognition and signaling events. The major histocompatibility complex (MHC) class I homolog MIC-A is a stress-inducible self antigen that is broadly recognized by NK cells, CD8+ alpha beta T cells and the V51-bearing subset of gamma/delta T cells independent of beta2-microglobulin and bound peptides. Consequently, MIC-A has been proposed to "play an important role in the surveillance of transformed, infected and damaged cells" by the innate immune system in intestinal epithelium and epithelially derived tumors. MIC-A recognition by these cells is mediated through interactions with the stimulatory NK receptor NKG2D, a divergent member of the C-type lectin-like protein family and a distant relative of other members of the NKG2 family of NK cell receptors and CD94. Our recent crystallographic analysis of the three-dimensional structure of MIC-A reveals a dramatically altered MHC class I fold, both in detail and overall domain organization. We propose to characterize the biochemical and structural details of the interaction between the NK cell receptor NKG2D and the MIC family of proteins. This knowledge will allow us to understand an important NK recognition event, mediated by NKG2D, whose target is clearly unlike that of alpha beta T cell receptors or other NK cell receptors which recognize more conventional MHC class I proteins and homologs. We will achieve this goal by 1) expressing soluble forms of the extracellular domains of a series of members of the MIC protein family and NKG2D, using multiple expression systems; 2) determining binding affinities by both qualitative and quantitative assays; 3) delineating a proposed interaction site on MIC-A by site-directed mutational analysis; and 4) crystallizing and determining the three-dimensional structures of individual proteins and appropriate complexes by x-ray crystallography. We will also 5) determine the effect glycosylation has on i) the MIC/NKG2D interaction; ii) the stability of these proteins; and iii) their structures. Since the initial submission of this application, we have crystallized three of the species we propose to study, including the MIC-AfNKG2D complex, and have collected preliminary SPR binding data for one interaction (baculovirus-expressed MIC-A and bacterially-expressed NKG2D).